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1.  Autophagy-Dependent Metabolic Reprogramming Sensitizes TSC2-Deficient Cells to the Antimetabolite 6-Aminonicotinamide 
The mammalian target of rapamycin complex 1 (mTORC1) is hyperactive in many human cancers and in tuberous sclerosis complex (TSC). Autophagy, a key mTORC1 targeted process, is a critical determinant of metabolic homeostasis. Metabolomic profiling was performed to elucidate the cellular consequences of autophagy dysregulation under conditions of hyperactive mTORC1. It was discovered that TSC2-null cells have distinctive autophagy-dependent pentose phosphate pathway (PPP) alterations. This was accompanied by enhanced glucose uptake and utilization, decreased mitochondrial oxygen consumption, and increased mitochondrial ROS production. Importantly, these findings revealed that the PPP is a key autophagy-dependent compensatory metabolic mechanism. Furthermore, PPP inhibition with 6-aminonicotinamide (6-AN) in combination with autophagy inhibition suppressed proliferation and prompted the activation of NF-kB and CASP1 in TSC2-deficient, but not TSC2-proficient cells. These data demonstrate that TSC2-deficient cells can be therapeutically targeted, without mTORC1 inhibitors, by focusing on their metabolic vulnerabilities. Implications: This study provides proof-of-concept that therapeutic targeting of diseases with hyperactive mTORC1 can be achieved without the application of mTORC1 inhibitors.
doi:10.1158/1541-7786.MCR-13-0258-T
PMCID: PMC4030750  PMID: 24296756
2.  Tsc1-Tp53 loss induces mesothelioma in mice, and evidence for this mechanism in human mesothelioma 
Oncogene  2013;33(24):3151-3160.
Mesothelioma is diagnosed in approximately 2,500 patients in the United States every year, most often arising in the pleural space, but also occurring as primary peritoneal mesothelioma. The vast majority of patients with mesothelioma die from their disease within 3 years. We developed a new mouse model of mesothelioma by bladder or intra-peritoneal injection of adenovirus Cre into mice with conditional alleles of each of Tp53 and Tsc1. Such mice began to develop malignant ascites about 6 months after injection, which was due to peritoneal mesothelioma, based on tumor morphology and immunohistochemical staining. Mesothelioma cell lines were established which showed loss of both Tsc1 and Tp53, with mTORC1 activation. Treatment of mice with malignant ascites due to mesothelioma with rapamycin led to a marked reduction in ascites, extended survival, and a 95–99% reduction in mesothelioma tumor volume, in comparison to vehicle-treated mice. To see if TSC1/TSC2 loss was a common genetic event in human mesothelioma, we examined 9 human mesothelioma cell lines, and found that 4 of 9 showed persistent activation of mTORC1 though none had loss of TSC1 or TSC2. A tissue microarray analysis of 198 human mesothelioma specimens showed that 33% of cases had reduced TSC2 expression and 60% showed activation of mTOR, indicating that mTOR activation is common in human mesothelioma and suggesting that it is a potential therapeutic target.
doi:10.1038/onc.2013.280
PMCID: PMC3931745  PMID: 23851502
TSC1; TSC2; mesothelioma; rapamycin; mTOR
3.  Using Multiplexed Assays of Oncogenic Drivers in Lung Cancers to Select Targeted Drugs 
IMPORTANCE
Targeting oncogenic drivers (genomic alterations critical to cancer development and maintenance) has transformed the care of patients with lung adenocarcinomas. The Lung Cancer Mutation Consortium was formed to perform multiplexed assays testing adenocarcinomas of the lung for drivers in 10 genes to enable clinicians to select targeted treatments and enroll patients into clinical trials.
OBJECTIVES
To determine the frequency of oncogenic drivers in patients with lung adenocarcinomas and to use the data to select treatments targeting the identified driver(s) and measure survival.
DESIGN, SETTING, AND PARTICIPANTS
From 2009 through 2012, 14 sites in the United States enrolled patients with metastatic lung adenocarcinomas and a performance status of 0 through 2 and tested their tumors for 10 drivers. Information was collected on patients, therapies, and survival.
INTERVENTIONS
Tumors were tested for 10 oncogenic drivers, and results were used to select matched targeted therapies.
MAIN OUTCOMES AND MEASURES
Determination of the frequency of oncogenic drivers, the proportion of patients treated with genotype-directed therapy, and survival.
RESULTS
From 2009 through 2012, tumors from 1007 patients were tested for at least 1 gene and 733 for 10 genes (patients with full genotyping). An oncogenic driver was found in 466 of 733 patients (64%). Among these 733 tumors, 182 tumors (25%) had the KRAS driver; sensitizing EGFR, 122 (17%); ALK rearrangements, 57 (8%); other EGFR, 29 (4%); 2 or more genes, 24 (3%); ERBB2 (formerly HER2), 19 (3%); BRAF, 16 (2%); PIK3CA, 6 (<1%); MET amplification, 5 (<1%); NRAS, 5 (<1%); MEK1, 1 (<1%); AKT1, 0. Results were used to select a targeted therapy or trial in 275 of 1007 patients (28%). The median survival was 3.5 years (interquartile range [IQR], 1.96-7.70) for the 260 patients with an oncogenic driver and genotype-directed therapy compared with 2.4 years (IQR, 0.88-6.20) for the 318 patients with any oncogenic driver(s) who did not receive genotype-directed therapy (propensity score–adjusted hazard ratio, 0.69 [95% CI, 0.53-0.9], P = .006).
CONCLUSIONS AND RELEVANCE
Actionable drivers were detected in 64% of lung adenocarcinomas. Multiplexed testing aided physicians in selecting therapies. Although individuals with drivers receiving a matched targeted agent lived longer, randomized trials are required to determine if targeting therapy based on oncogenic drivers improves survival.
doi:10.1001/jama.2014.3741
PMCID: PMC4163053  PMID: 24846037
4.  Rapamycin-Insensitive Up-Regulation of Adipocyte Phospholipase A2 in Tuberous Sclerosis and Lymphangioleiomyomatosis 
PLoS ONE  2014;9(10):e104809.
Tuberous sclerosis syndrome (TSC) is an autosomal dominant tumor suppressor gene syndrome affecting multiple organs, including renal angiomyolipomas and pulmonary lymphangioleiomyomatosis (LAM). LAM is a female-predominant interstitial lung disease characterized by the progressive cyst formation and respiratory failure, which is also seen in sporadic patients without TSC. Mutations in TSC1 or TSC2 cause TSC, result in hyperactivation of mammalian target of rapamycin (mTOR), and are also seen in LAM cells in sporadic LAM. We recently reported that prostaglandin biosynthesis and cyclooxygenase-2 were deregulated in TSC and LAM. Phospholipase A2 (PLA2) is the rate-limiting enzyme that catalyzes the conversion of plasma membrane phospholipids into prostaglandins. In this study, we identified upregulation of adipocyte AdPLA2 (PLA2G16) in LAM nodule cells using publicly available expression data. We showed that the levels of AdPLA2 transcript and protein were higher in LAM lungs compared with control lungs. We then showed that TSC2 negatively regulates the expression of AdPLA2, and loss of TSC2 is associated with elevated production of prostaglandin E2 (PGE2) and prostacyclin (PGI2) in cell culture models. Mouse model studies also showed increased expression of AdPLA2 in xenograft tumors, estrogen-induced lung metastatic lesions of Tsc2 null leiomyoma-derived cells, and spontaneous renal cystadenomas from Tsc2+/− mice. Importantly, rapamycin treatment did not affect the expression of AdPLA2 and the production of PGE2 by TSC2-deficient mouse embryonic fibroblast (Tsc2−/−MEFs), rat uterine leiomyoma-derived ELT3 cells, and LAM patient-associated renal angiomyolipoma-derived “mesenchymal” cells. Furthermore, methyl arachidonyl fluorophosphate (MAFP), a potent irreversible PLA2 inhibitor, selectively suppressed the growth and induced apoptosis of TSC2-deficient LAM patient-derived cells relative to TSC2-addback cells. Our findings suggest that AdPLA2 plays an important role in promoting tumorigenesis and disease progression by modulating the production of prostaglandins and may serve as a potential therapeutic target in TSC and LAM.
doi:10.1371/journal.pone.0104809
PMCID: PMC4210122  PMID: 25347447
5.  Integrative analysis of 1q23.3 copy number gain in metastatic urothelial carcinoma 
Purpose
Metastatic urothelial carcinoma (UC) of the bladder is associated with multiple somatic copy number alterations (SCNAs). We evaluated SCNAs to identify predictors of poor survival in patients with metastatic UC treated with platinum-based chemotherapy.
Experimental Design
We obtained overall survival (OS) and array DNA copy number data from metastatic UC patients in two cohorts. Associations between recurrent SCNAs and OS were determined by a Cox proportional hazard model adjusting for performance status and visceral disease. mRNA expression was evaluated for potential candidate genes by Nanostring nCounter to identify transcripts from the region that are associated with copy number gain. In addition, expression data from an independent cohort was used to identify candidate genes.
Results
Multiple areas of recurrent significant gains and losses were identified. Gain of 1q23.3 was independently associated with a shortened OS in the both cohorts (adjusted HR 2.96; 95% CI, 1.35 to 6.48; P = 0.01 and adjusted HR 5.03; 95% CI 1.43-17.73; P < 0.001). The F11R, PFDN2, PPOX, USP21 and DEDD genes, all located on 1q23.3, were closely associated with poor outcome.
Conclusions
1q23.3 copy number gain displayed association with poor survival in two cohorts of metastatic UC. The identification of the target of this copy number gain is ongoing, and exploration of this finding in other disease states may be useful for the early identification of poor risk UC patients. Prospective validation of the survival association is necessary to demonstrate clinical relevance.
doi:10.1158/1078-0432.CCR-13-0759
PMCID: PMC3975677  PMID: 24486590
6.  Molecular Testing Guideline for Selection of Lung Cancer Patients for EGFR and ALK Tyrosine Kinase Inhibitors 
Objective
To establish evidence-based recommendations for the molecular analysis of lung cancers that are required to guide EGFR- and ALK-directed therapies, addressing which patients and samples should be tested, and when and how testing should be performed.
Participants
Three cochairs without conflicts of interest were selected, one from each of the 3 sponsoring professional societies: College of American Pathologists, International Association for the Study of Lung Cancer, and Association for Molecular Pathology. Writing and advisory panels were constituted from additional experts from these societies.
Evidence
Three unbiased literature searches of electronic databases were performed to capture articles published from January 2004 through February 2012, yielding 1533 articles whose abstracts were screened to identify 521 pertinent articles that were then reviewed in detail for their relevance to the recommendations. Evidence was formally graded for each recommendation.
Consensus Process
Initial recommendations were formulated by the cochairs and panel members at a public meeting. Each guideline section was assigned to at least 2 panelists. Drafts were circulated to the writing panel (version 1), advisory panel (version 2), and the public (version 3) before submission (version 4).
Conclusions
The 37 guideline items address 14 subjects, including 15 recommendations (evidence grade A/B). The major recommendations are to use testing for EGFR mutations and ALK fusions to guide patient selection for therapy with an epidermal growth factor receptor (EGFR) or anaplastic lymphoma kinase (ALK) inhibitor, respectively, in all patients with advanced-stage adenocarcinoma, regardless of sex, race, smoking history, or other clinical risk factors, and to prioritize EGFR and ALK testing over other molecular predictive tests. As scientific discoveries and clinical practice outpace the completion of randomized clinical trials, evidence-based guidelines developed by expert practitioners are vital for communicating emerging clinical standards. Already, new treatments targeting genetic alterations in other, less common driver oncogenes are being evaluated in lung cancer, and testing for these may be addressed in future versions of these guidelines.
doi:10.5858/arpa.2012-0720-OA
PMCID: PMC4162344  PMID: 23551194
7.  Molecular Testing Guideline for Selection of Lung Cancer Patients for EGFR and ALK Tyrosine Kinase Inhibitors 
Objective
To establish evidence-based recommendations for the molecular analysis of lung cancers that are that are required to guide EGFR- and ALK-directed therapies, addressing which patients and samples should be tested, and when and how testing should be performed.
Participants
Three cochairs without conflicts of interest were selected, one from each of the 3 sponsoring professional societies: College of American Pathologists, International Association for the Study of Lung Cancer, and Association for Molecular Pathology. Writing and advisory panels were constituted from additional experts from these societies.
Evidence
Three unbiased literature searches of electronic databases were performed to capture articles published published from January 2004 through February 2012, yielding 1533 articles whose abstracts were screened to identify 521 pertinent articles that were then reviewed in detail for their relevance to the recommendations. Evidence was formally graded for each recommendation.
Consensus Process
Initial recommendations were formulated by the cochairs and panel members at a public meeting. Each guideline section was assigned to at least 2 panelists. Drafts were circulated to the writing panel (version 1), advisory panel (version 2), and the public (version 3) before submission (version 4).
Conclusions
The 37 guideline items address 14 subjects, including 15 recommendations (evidence grade A/B). The major recommendations are to use testing for EGFR mutations and ALK fusions to guide patient selection for therapy with an epidermal growth factor receptor (EGFR) or anaplastic lymphoma kinase (ALK) inhibitor, respectively, in all patients with advanced-stage adenocarcinoma, regardless of sex, race, smoking history, or other clinical risk factors, and to prioritize EGFR and ALK testing over other molecular predictive tests. As scientific discoveries and clinical practice outpace the completion of randomized clinical trials, evidence-based guidelines developed by expert practitioners are vital for communicating emerging clinical standards. Already, new treatments targeting genetic alterations in other, less common driver oncogenes are being evaluated in lung cancer, and testing for these may be addressed in future versions of these guidelines.
doi:10.1097/JTO.0b013e318290868f
PMCID: PMC4159960  PMID: 23552377
8.  Metabolic and Functional Genomic Studies Identify Deoxythymidylate Kinase as a target in LKB1 Mutant Lung Cancer 
Cancer discovery  2013;3(8):870-879.
The LKB1/STK11 tumor suppressor encodes a serine/threonine kinase which coordinates cell growth, polarity, motility, and metabolism. In non-small cell lung cancer, LKB1 is somatically inactivated in 25-30% of cases, often concurrently with activating KRAS mutation. Here, we employed an integrative approach to define novel therapeutic targets in KRAS-driven LKB1 mutant lung cancers. High-throughput RNAi screens in lung cancer cell lines from genetically engineered mouse models driven by activated KRAS with or without coincident Lkb1 deletion led to the identification of Dtymk, encoding deoxythymidylate kinase which catalyzes dTTP biosynthesis, as synthetically lethal with Lkb1 deficiency in mouse and human lung cancer lines. Global metabolite profiling demonstrated that Lkb1-null cells had striking decreases in multiple nucleotide metabolites as compared to the Lkb1-wt cells. Thus, LKB1 mutant lung cancers have deficits in nucleotide metabolism conferring hypersensitivity to DTYMK inhibition, suggesting that DTYMK is a potential therapeutic target in this aggressive subset of tumors.
doi:10.1158/2159-8290.CD-13-0015
PMCID: PMC3753578  PMID: 23715154
LKB1; KRAS; DTYMK; CHEK1; NSCLC; GEMM-derived cell line; genome wide RNAi screen; metabolic profiling
9.  Estradiol and mTORC2 cooperate to enhance prostaglandin biosynthesis and tumorigenesis in TSC2-deficient LAM cells 
Estradiol enhances COX-2 expression and prostaglandin biosynthesis in TSC2-deficient cells via a rapamycin-insensitive, mTORC2-dependent mechanism.
Lymphangioleiomyomatosis (LAM) is a progressive neoplastic disorder that leads to lung destruction and respiratory failure primarily in women. LAM is typically caused by tuberous sclerosis complex 2 (TSC2) mutations resulting in mTORC1 activation in proliferative smooth muscle–like cells in the lung. The female predominance of LAM suggests that estradiol contributes to disease development. Metabolomic profiling identified an estradiol-enhanced prostaglandin biosynthesis signature in Tsc2-deficient (TSC−) cells, both in vitro and in vivo. Estradiol increased the expression of cyclooxygenase-2 (COX-2), a rate-limiting enzyme in prostaglandin biosynthesis, which was also increased at baseline in TSC-deficient cells and was not affected by rapamycin treatment. However, both Torin 1 treatment and Rictor knockdown led to reduced COX-2 expression and phospho-Akt-S473. Prostaglandin production was also increased in TSC-deficient cells. In preclinical models, both Celecoxib and aspirin reduced tumor development. LAM patients had significantly higher serum prostaglandin levels than healthy women. 15-epi-lipoxin-A4 was identified in exhaled breath condensate from LAM subjects and was increased by aspirin treatment, indicative of functional COX-2 expression in the LAM airway. In vitro, 15-epi-lipoxin-A4 reduced the proliferation of LAM patient–derived cells in a dose-dependent manner. Targeting COX-2 and prostaglandin pathways may have therapeutic value in LAM and TSC-related diseases, and possibly in other conditions associated with mTOR hyperactivation.
doi:10.1084/jem.20131080
PMCID: PMC3892971  PMID: 24395886
10.  Equivalent benefit of rapamycin and a potent mTOR ATP-competitive inhibitor, MLN0128 (INK128), in a mouse model of tuberous sclerosis 
Molecular cancer research : MCR  2013;11(5):467-473.
Tuberous sclerosis complex (TSC) is a hamartoma syndrome in which brain, renal and lung tumors develop and cause both morbidity and death. Loss of either TSC1 or TSC2 in TSC hamartomas leads to activation of mTORC1. Rapamycin and related drugs have been shown to have clinical benefit for these tumors in TSC patients and those with sporadic forms of TSC-related neoplasms. However, lifelong therapy appears to be required, as tumors are not eliminated by this treatment. We examined the potential benefit of MLN0128, a novel potent mTOR ATP-competitive inhibitor, as a therapeutic strategy for renal cystadenomas that develop in A/J Tsc2+/− mice. Rapamycin given by intraperitoneal injection at 3 mg/kg 3 times per week, and MLN0128 given by gavage at 0.75 mg/kg 5 times per week had equivalent effects in suppressing tumor development during a 4 week treatment period, with an approximate 99% reduction in microscopic tumor cell volume. Marked reduction in activation of mTORC1, and blockade of cell growth was seen with both drugs, while only MLN0128 treatment had effects in blocking mTORC2 and 4EBP1 phosphorylation. However, when either drug was discontinued and mice were observed for two additional months, there was dramatic recovery of tumor growth, with extensive proliferation. Hence, long-lasting tumor growth control is not achieved with transient treatment with either drug, and MLN0128 and rapamycin have equivalent therapeutic benefit in this mouse model. Differences in side-effect profiles might make MLN0128 more attractive for treatment of patients with TSC-related tumors, but will require additional study in humans.
doi:10.1158/1541-7786.MCR-12-0605
PMCID: PMC3657392  PMID: 23386687
TSC; mTOR; INK128; rapamycin; MLN0128
11.  Design and Generation of MLPA Probe Sets for Combined Copy Number and Small-Mutation Analysis of Human Genes: EGFR as an Example 
TheScientificWorldJournal  2010;10:2003-2018.
Multiplex ligation-dependent probe amplification (MLPA) is a multiplex copy number analysis method that is routinely used to identify large mutations in many clinical and research labs. One of the most important drawbacks of the standard MLPA setup is a complicated, and therefore expensive, procedure of generating long MLPA probes. This drawback substantially limits the applicability of MLPA to those genomic regions for which ready-to-use commercial kits are available. Here we present a simple protocol for designing MLPA probe sets that are composed entirely of short oligonucleotide half-probes generated through chemical synthesis. As an example, we present the design and generation of an MLPA assay for parallel copy number and small-mutation analysis of the EGFR gene.
doi:10.1100/tsw.2010.195
PMCID: PMC4004796  PMID: 20953551
multiplex ligation-dependent probe amplification; MLPA; copy number variation; CNV; EGFR; large deletion; amplification; mutation detection
12.  Extrarenal perivascular epithelioid cell tumors (PEComas) respond to mTOR inhibition: Clinical and molecular correlates 
Perivascular epithelioid cell tumors (PEComas) are a group of rare mesenchymal tumors that typically show both melanocytic and smooth muscle cell features. Some types of PEComa are seen at high frequency in tuberous sclerosis complex (TSC). The TSC1 and TSC2 genes are commonly mutated in both TSC-associated and sporadic PEComas, and mTOR signaling pathway activation is also common in these tumors. Preliminary reports have indicated that the mTOR inhibitors sirolimus and related drugs have activity in some patients with non-TSC-associated PEComa.
Here we report on the use of these medications in the treatment of five consecutive patients with extrarenal non-pulmonary PEComas seen at one institution. Three complete responses, one partial response and one case of progression were seen. Molecular studies identified TSC2 aberrations in four of these patients, and TFE3 translocation was excluded in the resistant case. A review of all published cases as well as those reported here indicates that partial or complete response was seen in 6 of 11 PEComas, with 5 of the 6 having a complete response. These findings highlight the consistent though incomplete activity of mTOR inhibitors in the treatment of PEComas.
doi:10.1002/ijc.27800
PMCID: PMC3558545  PMID: 22927055
perivascular epithelioid cell tumor; PEComa; mTOR; TSC2; sirolimus; everolimus
14.  Molecular dissection of AKT activation in lung cancer cell lines 
Molecular cancer research : MCR  2013;11(3):282-293.
AKT is a critical signaling node downstream of PI3K, which is often activated in cancer. We analyzed the state of activation of AKT in 80 human non-small cell lung cancer cell lines under serum starvation conditions. We identified 13 lines which showed persistent AKT activation in the absence of serum. In 12 of the 13 lines, AKT activation could be attributed to loss of PTEN, activating mutation in EGFR or PIK3CA, or amplification of ERBB2. HCC2429 was the only cell line that had no alterations in those genes, but had high phospho-AKT(Ser473) levels under serum starvation conditions. However, the activation of AKT in HCC2429 was PI3K- and mTORC2-dependent based upon use of specific inhibitors. Kinome tyrosine phosphorylation profiling showed that both Notch and SRC were highly activated in this cell line. Despite the activation of Notch, AKT activation and cell survival were not affected by Notch inhibitors DAPT or Compound E. In contrast, SRC inhibitors PP2 and dasatinib both significantly decreased pAKT(Ser473) levels and reduced cell survival by inducing apoptosis. Further, a combination of SRC and mTOR inhibition synergistically blocked activation of AKT and induced apoptosis. Over-expression of SRC has been identified previously in human lung cancers, and these results suggest that a combination of SRC and mTOR inhibitors may have unique therapeutic benefit for a subset of lung cancers with these molecular features.
doi:10.1158/1541-7786.MCR-12-0558
PMCID: PMC3606262  PMID: 23319332
lung cancer; AKT; mTOR; SRC; rapamycin; Torin1
15.  Prenatal rapamycin results in early and late behavioral abnormalities in wildtype C57Bl/6 mice 
Behavior genetics  2012;43(1):51-59.
Mammalian target of rapamycin (mTOR) signaling has been shown to be deregulated in a number of genetic, neurodevelopmental disorders including Tuberous Sclerosis Complex, Neurofibromatosis, Fragile X, and Rett syndromes. As a result, mTOR inhibitors, such as rapamycin and its analogs, offer potential therapeutic avenues for these disorders. Some of these disorders – such as Tuberous Sclerosis Complex – can be diagnosed prenatally. Thus, prenatal administration of these inhibitors could potentially prevent the development of the devastating symptoms associated with these disorders. To assess the possible detrimental effects of prenatal rapamycin treatment, we evaluated both early and late behavioral effects of a single rapamycin treatment at embryonic day 16.5 in wildtype C57Bl/6 mice. This treatment adversely impacted early developmental milestones as well as motor function in adult animals. Rapamycin also resulted in anxiety-like behaviors during both early development and adulthood but did not affect adult social behaviors. Together, these results indicate that a single, prenatal rapamycin treatment not only adversely affects early postnatal development but also results in long lasting negative effects, persisting into adulthood. These findings are of importance in considering prenatal administration of rapamycin and related drugs in the treatment of patients with neurogenetic, neurodevelopmental disorders.
doi:10.1007/s10519-012-9571-9
PMCID: PMC3554236  PMID: 23229624
mTOR; tuberous sclerosis; embryonic; mouse
16.  The introduction of systematic genomic testing for patients with non-small cell lung cancer 
Background
Genomic testing to identify driver mutations that enable targeted therapy is emerging for patients with non-small cell lung cancer (NSCLC). We report the implementation of systematic prospective genotyping for somatic alterations in BRAF, PIK3CA, HER2, and ALK, in addition to EGFR and KRAS, in NSCLC patients at the Dana-Farber Cancer Institute.
Methods
Patients with NSCLC were prospectively referred by their providers for clinical genotyping. Formalin-fixed, paraffin embedded tumor samples were analyzed by Sanger sequencing for mutations in selected exons of EGFR, KRAS, BRAF, PIK3CA, and HER2. ALK rearrangements were detected by FISH or immunohistochemistry.
Results
Between 7/1/2009 and 8/1/2010, 427 specimens from 419 patients were referred for genomic characterization; 344 (81%) specimens were successfully genotyped with a median turnaround time of 31 days (range, 9-155). Of the 344 specimens, 185 (54%) had at least one identifiable somatic alteration (KRAS: 24%, EGFR: 17%, ALK: 5%, BRAF: 5%, HER2: 4%, PIK3CA: 2%). As of 8/1/2011, 63/288 (22%) advanced NSCLC patients had received molecularly targeted therapy based on their genotypic results, including 34/42 (81%) patients with EGFR mutations, 12/15 (80%) with ALK rearrangements, and 17/95 (18%) with KRAS, BRAF or HER2 mutations.
Conclusions
Large scale testing for somatic alterations in EGFR, KRAS, BRAF, PIK3CA, HER2 and ALK is feasible and impacts therapeutic decisions. As the repertoire for personalized therapies expands in lung cancer and other malignancies, there is a need to develop new genomics technologies that can generate a comprehensive genetic profile of tumor specimens in a time and cost effective manner.
doi:10.1097/JTO.0b013e3182745bcb
PMCID: PMC3500523  PMID: 23154547
Lung cancer; cancer genomics; molecular targeted therapy
17.  Correction: Stochastic Model of Tsc1 Lesions in Mouse Brain 
PLoS ONE  2013;8(11):10.1371/annotation/6a5b0a50-27e4-49bc-b82a-9267dd63af53.
doi:10.1371/annotation/6a5b0a50-27e4-49bc-b82a-9267dd63af53
PMCID: PMC3821742  PMID: 24244248
18.  Graded loss of tuberin in an allelic series of brain models of TSC correlates with survival, and biochemical, histological and behavioral features 
Human Molecular Genetics  2012;21(19):4286-4300.
Tuberous sclerosis complex (TSC) is a neurodevelopmental disorder with prominent brain manifestations due to mutations in either TSC1 or TSC2. Here, we describe novel mouse brain models of TSC generated using conditional hypomorphic and null alleles of Tsc2 combined with the neuron-specific synapsin I cre (SynIcre) allele. This allelic series of homozygous conditional hypomorphic alleles (Tsc2c-del3/c-del3SynICre+) and heterozygote null/conditional hypomorphic alleles (Tsc2k/c-del3SynICre+) achieves a graded reduction in expression of Tsc2 in neurons in vivo. The mice demonstrate a progressive neurologic phenotype including hunchback, hind limb clasp, reduced survival and brain and cortical neuron enlargement that correlates with a graded reduction in expression of Tsc2 in the two sets of mice. Both models also showed behavioral abnormalities in anxiety, social interaction and learning assays, which correlated with Tsc2 protein levels as well. The observations demonstrate that there are graded biochemical, cellular and clinical/behavioral effects that are proportional to the extent of reduction in Tsc2 expression in neurons. Further, they suggest that some patients with milder manifestations of TSC may be due to persistent low-level expression of functional protein from their mutant allele. In addition, they point to the potential clinical benefit of strategies to raise TSC2 protein expression from the wild-type allele by even modest amounts.
doi:10.1093/hmg/dds262
PMCID: PMC3441124  PMID: 22752306
19.  Mapping the hallmarks of lung adenocarcinoma with massively parallel sequencing 
Cell  2012;150(6):1107-1120.
SUMMARY
Lung adenocarcinoma, the most common subtype of non-small cell lung cancer, is responsible for over 500,000 deaths per year worldwide. Here, we report exome and genome sequences of 183 lung adenocarcinoma tumor/normal DNA pairs. These analyses revealed a mean exonic somatic mutation rate of 12.0 events/megabase and identified the majority of genes previously reported as significantly mutated in lung adenocarcinoma. In addition, we identified statistically recurrent somatic mutations in the splicing factor gene U2AF1 and truncating mutations affecting RBM10 and ARID1A. Analysis of nucleotide context-specific mutation signatures grouped the sample set into distinct clusters that correlated with smoking history and alterations of reported lung adenocarcinoma genes. Whole genome sequence analysis revealed frequent structural re-arrangements, including in-frame exonic alterations within EGFR and SIK2 kinases. The candidate genes identified in this study are attractive targets for biological characterization and therapeutic targeting of lung adenocarcinoma.
doi:10.1016/j.cell.2012.08.029
PMCID: PMC3557932  PMID: 22980975
20.  TBC1D7 is a third subunit of the TSC1-TSC2 complex upstream of mTORC1 
Molecular cell  2012;47(4):535-546.
SUMMARY
The tuberous sclerosis complex (TSC) tumor suppressors form the TSC1-TSC2 complex, which limits cell growth in response to poor growth conditions. Through its GTPase-activating protein (GAP) activity toward Rheb, this complex inhibits the mechanistic target of rapamycin (mTOR) complex 1 (mTORC1), a key promoter of cell growth. Here, we identify and biochemically characterize TBC1D7 as a stably-associated and ubiquitous third core subunit of the TSC1-TSC2 complex. We demonstrate that the TSC1-TSC2-TBC1D7 (TSC-TBC) complex is the functional complex that senses specific cellular growth conditions and possesses Rheb-GAP activity. Sequencing analyses of samples from TSC patients suggest that TBC1D7 is unlikely to represent TSC3. TBC1D7 knockdown decreases the association of TSC1 and TSC2 leading to decreased Rheb-GAP activity, without effects on the localization of TSC2 to the lysosome. Like the other TSC-TBC components, TBC1D7 knockdown results in increased mTORC1 signaling, delayed induction of autophagy, and enhanced cell growth under poor growth conditions.
doi:10.1016/j.molcel.2012.06.009
PMCID: PMC3693578  PMID: 22795129
21.  Chronic Activation of mTOR Complex 1 is Sufficient to Cause Hepatocellular Carcinoma 
Science signaling  2012;5(217):ra24.
The mammalian target of rapamycin (mTOR) complex 1 (mTORC1) is a nutrient sensitive protein kinase that is aberrantly activated in many human cancers. However, whether dysregulation of mTORC1 signaling in normal tissues contributes to cancer risk is unknown. Here, we focused on hepatocellular carcinoma because it is a cancer with clear links to environmental factors that affect mTORC1, including dietary influences. Genetic ablation of the mTORC1 inhibitory component Tsc1 results in constitutively elevated mTORC1 signaling, an effect similar to that of obesity on this pathway. We found that mice with liver-specific knockout of Tsc1 developed sporadic hepatocellular carcinoma with heterogeneous histological and biochemical features. The spontaneous development of hepatocellular carcinoma in this mouse model was preceded by a series of pathological changes known to accompany the primary etiologies of this cancer, including liver damage, inflammation, necrosis, and regeneration. Chronic mTORC1 signaling caused unresolved endoplasmic reticulum stress and defects in autophagy, which contributed to hepatocyte damage and hepatocellular carcinoma development. Therefore, we demonstrate a previously unrecognized role for mTORC1 in carcinogenesis, perhaps representing a key molecular link between cancer risk and environmental factors, such as diet.
doi:10.1126/scisignal.2002739
PMCID: PMC3743103  PMID: 22457330
22.  IL10 Gene Polymorphisms Are Associated With Asthma Phenotypes in Children 
Genetic epidemiology  2004;26(2):155-165.
IL10 is an anti-inflammatory cytokine that has been found to have lower production in macrophages and mononuclear cells from asthmatics. Since reduced IL10 levels may influence the severity of asthma phenotypes, we examined IL10 single-nucleotide polymorphisms (SNPs) for association with asthma severity and allergy phenotypes as quantitative traits. Utilizing DNA samples from 518 Caucasian asthmatic children from the Childhood Asthma Management Program (CAMP) and their parents, we genotyped six IL10 SNPs: 3 in the promoter, 2 in introns, and one in the 3′ UTR. Using family-based association tests, each SNP was tested for association with asthma and allergy phenotypes individually. Population-based association analysis was performed with each SNP locus, the promoter haplotypes and the 6-loci haplotypes. The 3′ UTR SNP was significantly associated with FEV1 as a percent of predicted (FEV1PP) (P=0.0002) in both the family and population analyses. The promoter haplotype GCC was positively associated with IgE levels and FEV1PP (P=0.007 and 0.012, respectively). The promoter haplotype ATA was negatively associated with lnPC20 and FEV1PP (P=0.008 and 0.043, respectively). Polymorphisms in IL10 are associated with asthma phenotypes in this cohort. Further studies of variation in the IL10 gene may help elucidate the mechanism of asthma development in children.
doi:10.1002/gepi.10298
PMCID: PMC3705717  PMID: 14748015
interleukin 10 (IL10); single nucleotide polymorphism (SNP); genetic association; family-based association test (FBAT); haplotype; promoter; 3′; untranslated region (3′UTR)
23.  Leydig Cell Tumor of the Testis in Tuberous Sclerosis: Lack of Second Hit Events 
The Oncologist  2012;17(7):927-929.
This article presents the case of an adult patient with tuberous sclerosis complex who presented with large right benign and left malignant Leydig cell tumors. The tumors were examined to determine if they showed the classic hallmarks of TSC1/TSC2 involvement.
doi:10.1634/theoncologist.2012-0045
PMCID: PMC3399648  PMID: 22707517
Tuberous sclerosis; Leydig cell tumor; TSC1
24.  Stochastic Model of Tsc1 Lesions in Mouse Brain 
PLoS ONE  2013;8(5):e64224.
Tuberous sclerosis complex (TSC) is an autosomal dominant disorder due to mutations in either TSC1 or TSC2 that affects many organs with hamartomas and tumors. TSC-associated brain lesions include subependymal nodules, subependymal giant cell astrocytomas and tubers. Neurologic manifestations in TSC comprise a high frequency of mental retardation and developmental disorders including autism, as well as epilepsy. Here, we describe a new mouse model of TSC brain lesions in which complete loss of Tsc1 is achieved in multiple brain cell types in a stochastic pattern. Injection of an adeno-associated virus vector encoding Cre recombinase into the cerebral ventricles of mice homozygous for a Tsc1 conditional allele on the day of birth led to reduced survival, and pathologic findings of enlarged neurons, cortical heterotopias, subependymal nodules, and hydrocephalus. The severity of clinical and pathologic findings as well as survival was shown to be dependent upon the dose and serotype of Cre virus injected. Although several other models of TSC brain disease exist, this model is unique in that the pathology reflects a variety of TSC-associated lesions involving different numbers and types of cells. This model provides a valuable and unique addition for therapeutic assessment.
doi:10.1371/journal.pone.0064224
PMCID: PMC3655945  PMID: 23696872
25.  Akt stimulates hepatic SREBP1c and lipogenesis through parallel mTORC1-dependent and independent pathways 
Cell metabolism  2011;14(1):21-32.
Through unknown mechanisms, insulin activates the sterol regulatory element-binding protein (SREBP1c) transcription factor to promote hepatic lipogenesis. We find that this induction is dependent on the mammalian target of rapamycin (mTOR) complex 1 (mTORC1). To further define the role of mTORC1 in the regulation of SREBP1c in the liver, we generated mice with liver-specific deletion of TSC1 (LTsc1KO), which results in insulin-independent activation of mTORC1. Surprisingly, the LTsc1KO mice are protected from age- and diet-induced hepatic steatosis and display hepatocyte-intrinsic defects in SREBP1c activation and de novo lipogenesis. These phenotypes result from attenuation of Akt signaling driven by mTORC1-dependent insulin resistance. Therefore, mTORC1 activation is not sufficient to stimulate hepatic SREBP1c in the absence of Akt signaling, revealing the existence of an additional downstream pathway also required for this induction. We provide evidence that this mTORC1-independent pathway involves Akt-mediated suppression of Insig2a, a liver-specific transcript encoding the SREBP1c inhibitor INSIG2.
doi:10.1016/j.cmet.2011.06.002
PMCID: PMC3652544  PMID: 21723501

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